The first project area explores metabolic pathways that have been proposed based on in vitro studies to be important in non-replicating (NR)-MTb. We are exploring the importance of biotin and pyridoxal biosynthesis, peptidoglycan turnover, the role of putative F420-binding and genetically annotated pyridoxal-generating enzymes, beta-oxidation and iron acquisition and validating these by chemical and genetic means in NR-MTb. We have shown that Rv2607 is the canonical pyridoxine phosphate oxidase of MTb and have enzymatically characterized this enzyme. In contrast, Rv1155, which is also annotated as a pyridoxine phosphate oxidase family protein has been expressed, purified and biophysically characterized and shown to bind the cofactor F420 and we are attempting to identify the natural substrate of this protein by fragment based screening approaches. We have also demonstrated the importance of biotin synthesis for the viability of MTb. Our studies of mycobacterial cell wall synthesis using meropenem as probe have allowed us to track the formation of the various layers of the mycobacterial cell wall during its assembly using a combination of cryo-electron, transmission and scanning electron microscopy. In addition, meropenem treatment of MTb is associated with a considerable fraction of phenotypically tolerant persisters. We are trying to understand the pathways that lead to a state of metabolic persistence as opposed to the pathways that are associated with cell death in order to identify targets of which inhibition would prevent the emergence of drug tolerant persisters or targets whose activation or inactivation could irreversibly set the course to a destiny of cell death. In this respect, we are collaborating with researchers at UC Berkeley who are characterizing the essential protein kinase B from MTb which is thought to be important for persistence and which contains potential beta-lactam binding domains which we have hypothesized may bind to muropeptides released during meropenem inhibition of its cell wall targets. The metabolic stresses that Mtb encounters in vivo may underscore the importance of certain metabolic adaptations that are critical for persistence inhibition of which may lead to death in vivo as opposed to stasis in vitro as exemplified by the in vivo cidal effect of pyrazinamide as well as linezolid, both of which are at best, static in vitro. Understanding these stresses are an important aspect of these studies. This has uncovered the role of the ribosome and the associated methylases that methylate ribosomal RNA in metabolic adaptation. The second major focus area of this project starts from a different perspective and uses compounds that are in clinical development (PA-824 and SQ109) which are known to possess activity against replicating as well as NR-TB. Various PA-824 analogs were analyzed for their activity with whole cell assays as well as assays with purified protein in order to further probe the substrate binding pocket of Dd, the F420-dependent reductase that activates PA824. We are attempting to understand what the natural substrate is for the Ddn, since this will allow us to probe the enzymatic processes that are important during non-replicating persistence. For SQ109 we have shown that inhibition of MTb with this compound results in a unique signature of cell wall metabolites where trehalose monomycolate accumulates with concomitant decrease of cell wall associated trehalose dimycolate suggesting inhibition of the trehalose monomycolate exporter. In accordance with this, mutations in the large transmembrane putative transporter mmpL3 gene, confer resistance to SQ109 and a variety of other hydrophobic scaffolds. To further unravel the key events in cell wall mycolyl-arabinogalactan synthesis, we have enzymatically characterized the three mycolyl transferase enzymes (Antigens 85 A, B and C) and are using a combination of site directed mutagenesis, regulated expression of GFP-Ag85 fusion enzymes in MTb and measurements of cellular mycolate synthesis to explore the functional importance of these homologs in MTb. We have found that the enzymes are kinetically distinct with Ag85C being enzymatically the most active and that certain amino acid residues outside of the substrate binding site in Ag85C, are important for catalytic activity. The third major focus of this project involves global approaches to understanding the metabolism in NR-TB. Using a chemostat model of MTb combined with metabolomic studies, we demonstrated that the NADH/NAD+ ratio changed as a function of oxygen concentration, that the direction of the TCA cycle reverses under hypoxia with concomitant extracellular succinate accumulation which is consistent with a model of oxygen-induced stasis in which an energized membrane is maintained by coupling the reductive branch of the TCA cycle to succinate secretion. An essential non-redundant step in this process is fumarase and we have initiated studies to identify fumarase inhibitors to validate the role of the forward as opposed to reverse TCA cycle in vitro as well as in vivo by using structure-based design based on the fumarase crystal structure to design inhibitors of this target. In a fourth approach, we are identifying inhibitors of metabolism by high-throughput screening approaches performed under a variety of in vivo relevant environmental conditions. Hits from these screens have provided a useful tool to map metabolism of MTb as a function of carbon source, oxygen concentration or presence of low pH in the presence or absence of nitrosative stress and are currently being studied to identify the target. In the process of target identification, parallel studies are done to rapidly progress the hits to in vivo proof of concept studies so that the importance of the target for in vivo pathogenesis can be validated early on in the drug discovery process. We are studying some of the hits that were identified from a 35,000 compound BioFocus collection in collaboration with various researchers in South Africa. The scaffolds that gave us evidence of a specific target based on SAR studies were taken further into target identification by a combination of approaches including resistant mutant generation followed by whole genome sequencing to identify single nucleotide polymorphisms, transcriptional profiling, macromolecular incorporation assays and metabolomics studies. For 2 chemically different scaffolds, the same target in mycobacterial cell wall synthesis was identified and we have initiated collaborative studies with researchers at the universities of Birmingham as well as Queensland to crystallize the target as well as explore the vulnerability of this enzyme. The targets of seven other scaffolds were identified. For several scaffolds, generation of resistant mutants was impossible and in several of these cases, inability to generate resistant mutants was correlated with mammalian cytotoxicity suggesting a non-specific mechanism of action. Resistance to another hit mapped to an enzyme in folate metabolism and we are currently attempting to characterize this enzyme as well as the associated enzymatic step prior to this by kinetic as well as crystallographic approaches. With collaborators at Weill Cornell Medical College, we have used this inhibitor as well as other known inhibitors of folate biosynthetic enzymes to explore which steps of this metabolic pathway are sensitive to perturbation. We are also studying the effect of changes in gene expression in cofactor metabolic pathways to identify how downregulation of cofactor biosynthesis affects target vulnerability. To date, we have completed a screen of MTb during restricted expression of panthotenate synthase which is essential for coenzyme A biosynthesis.